Powerpoint Assignment

Required Powerpoint Report (2017)

Reports Are Due Midnight Friday January 27, 2017.

You will not present your Powerpoint to the class. Your Powerpoint report will be graded, but it is not a test. It will simply be a record to document your practical work and understanding. You are encouraged to discuss your work with other students and ask for help from the instructors.
Email your Powerpoint files to martzeric@yahoo.com. (You may also use emartz@microbio.umass.edu but there may be problems sending attachments to that address.)
If you choose a homology model, email that homology model to me. It can be zipped. Or you can send me a dropbox link.

Important Instructions:

Use FirstGlance to answer all questions. Even if you know another method (such as PyMOL), I want you to be able to compare the experience in FirstGlance. It can be useful to add additional slides to show another method.
Use the model you selected for most of your report. Use your assigned PDB code only when the question asks you to do so. Do not use your assigned PDB code for your entire report.
Name your Powerpoint file with your family name, then first name: martzeric.ppt, kanejeffrey.pptx. You will earn 2 points extra credit if you follow this request.
Please show the Section Number on each slide.
Please show the PDB code used on each slide, or say homology model. You will lose points if you make me guess which model you are showing.
When the question says Snapshot, a snapshot is required. You can use "Save Image" built into FirstGlance, or you can save an animation. You must have at least one FirstGlance animation in your report (in addition to the PolyView-3D animation in Section 17).
You may make more than one slide to answer one question: "Section 1A", 1B, etc.
Extra work (for example by showing an additional model) or thoughtful comments may earn extra credit.

Examples of completed reports:

Example report for PDB code
Example report for homology model

Section 1 - Name, PDB codes

Your name.
Your email address.
Name of Professor of your laboratory research group.
Topic of your research project.

Model that you chose, either a PDB code or a homology model.
PDB code assigned to you.

PDB Code.
Name and function of the molecule.
Resolution, R value, and Free R of this model. FirstGlance.Jmol.Org gives R and Free R.
What "grade" does FirstGlance give to the Free R?

homology model

homology model sample slides

Generate your own homology model by submitting your sequence to Swiss-Model. This will give an up-to-date result and the best sequence alignment.
Before you copy the sequence alignment from SwissModel, click the small gray gear at the upper left corner of the sequence alignment. Then:
- Check "fade mismatches".
- Check "None" for secondary structure.
- Check "Indels" for color scheme. (You may have to click something else, then click Indels to get the colors right. See the example in the demo report./Users/emartz/Desktop/firstglance_image1.png )
- Export Alignment as PNG Image (to paste into your report).
In the sequence alignment, look for a gap in the model sequence that is not at either end (so embedded within the sequence) and 4 or more amino acids long. If such a gap (or gaps) exist, choose one and show its location in the homology model, using the example slide 1D as a guide.

Section 2 - Number of chains

Number of chains of protein, DNA, and RNA in the asymmetric unit (published PDB file). (A "chain" is a covalently linked linear polymer of amino acids or nucleotides. "Distinct" means the sequences are different.) Snapshot of Cartoon. If there are zero, say 0 chains of DNA, etc. Indicate which chains, if any, have identical sequences.
Suggestion: Copy the report from FirstGlance, Molecule Information Tab. including the Chain Details.

How to Make a Snapshot

When you need Ångstroms, copy this into Powerpoint: Å

Section 3 - Ligands and non-standard residues

Answer for both the model YOU chose, and the PDB code assigned to you:

What are the 3-letter codes, and full names of the ligands and non-standard residues?

Section 4 - Full length vs. crystallized sequence

For each protein chain, give the length of the crystallized fragment (number of amino acids), and the length of the full-length protein. Pick one chain and do a sequence alignment between the full-length protein and the crystallized fragment. Snapshot showing the alignment.
State some conclusions. For example, if the crystallized sequence is shorter than the full-length sequence, which parts were removed before crystallization: the C terminus, the N terminus, or both?
Suggestion: FirstGlance in Jmol, Molecule Information tab (PDB code tab), Sequences. At the bottom is a link to instructions for making the sequence alignment.

Section 5 - Secondary structure
(Use the model that YOU chose.)
What secondary structures are present? Is the molecule mostly one secondary structure? Animation or Snapshot.
Suggestion: FirstGlance in Jmol, Views tab, Secondary Structure.

Section 6 -- Charge distribution.

Are there any patches of all positive or all negative charges? Snapshot.
Choose a chain for charge calculations. Specify the one-letter name of the chain.
- For a PDB code, use the sequence of the crystallized protein fragment.
- For a homology model, use the full-length sequence of your protein for the pI/charge calcuation. If you would rather use a partial sequence, paste the sequence you used into your report so I can verify the numbers you report.
If you chose a PDB code, does the crystallized sequence include a poly-histidine tag? Does that affect the pI and charges?
What is the isoelectric pH (pI) of the chain?

Suggestion: FirstGlance, Views tab, Charge..

What is the charge of this chain at pH 4?
What is the charge of this chain at pH 7?
What is the charge of this chain at pH 10?

Section 7 - Local uncertainty

Display your molecule as sticks (Views tab, Vines, check more detail) then color by Local Uncertainty (Views tab).
List one residue with a high temperature (for example "Glu114 in chain B").
Put halos around the atoms of the listed residue with Find.
Show a snapshot including the minimum, average, and maximum temperature report at the upper right of Jmol. (To slide the molecule, hold down Ctrl and drag with the right mouse button.) Orient the molecule to show the region with highest temperature, and the halos on the listed residue.

Section 8 - Hydrophobic cores

Are there hydrophobic cores? Snapshot. (Don't just show a snapshot. Answer the question!) Note: there will be a separate hydrophobic core in each water soluble domain.
Suggestion: FirstGlance in Jmol, Views tab, Hydrophobic/Polar, turn on Slab.

Section 9 - Water solubility

Is your molecule soluble in water? Please say the reason for your conclusion. Snapshot.
Try the water toggle button in the Views or Tools tab. If your model has water, say what you think about the result.

FirstGlance in Jmol, Views tab, Hydrophobic/Polar

Section 10 - Disulfide bonds

If it has NO disulfide bonds, also show the PDB assigned to you.

Number of disulfide bonds? Number within chains? Between chains? ("Within" means from one Cys in a chain to another Cys in the same chain. "Between" means from a Cys in one chain to a Cys in a different chain.) Snapshot.
Suggestion: FirstGlance in Jmol, Tools tab, Disulfides. To distinguish within/between, check Color Disulfide Bonds By Chain.

Section 11 - Missing residues

TEMPLATE

If the PDB that YOU chose, or the TEMPLATE, has no missing residues, also do the ASSIGNED PDB code.

List missing residues. If there are none, say so.
Suggestion: In FirstGlance, Molecule Information Tab, Missing Residues.
Show a snapshot of the Summary by Chain table.
Show an animation or snapshot showing the "empty baskets".
How many incomplete amino acid sidechains? Suggestion: In FirstGlance, Molecule tab, the number of incomplete sidechains is given under Asymmetric Unit.

Section 12 - Non-covalent interactions

Use the model that you chose. If it has no ligand, you can target one amino acid, or if the PDB code assigned to you has ligand, you may use that.

Snapshots of close-ups of two different kinds of non-covalent bonds, using Contacts (Tools tab) in FirstGlance. Zoom in to show the bonds clearly.
Be sure to use the 4th Contacts display (balls and sticks colored by chemical element).
Show distances for only those two bonds.
Label the residues shown (e.g. Lys23:A, Glu44:B, where the letter after the colon is the chain name) so that your illustration can be confirmed for grading.
Say the kind (type, name) of each non-covalent bond you are showing.

Section 13 - Biological unit

template PDB code

Number of chains in the biological unit: two snapshots showing the asymmetric unit side by side with biological unit. Label each snapshot clearly and say the number of chains in each.
Suggestion: In FirstGlance, Resources tab, Biological Unit.

Section 14 - Evolutionary Conservation

ConSurfDB

If you chose a homology model, skip ConSurfDB and jump directly to step e below.
Enter your PDB code, and click the button Show Chains.
If there is more than one chain, choose one. Generally the longest chain will be most interesting.
Look for a section Main results, and click the button Structure colored by ConSurf results in FirstGlance.
If there are no results available at ConSurfDB, then submit your protein to the ConSurf Server. See the ConSurf Server Procedure at the bottom of this page.

After you have conservation in FirstGlance:

Find two highly conserved amino acids on the surface of your molecule, one expected and one unexpected. See (Why is conservation of Gly and Pro expected on the surface?)

Evolutionary Conservation
Expected	Unexpected
Functional site (patch of highly conserved amino acids) Gly in turn Pro* in turn Met1 at N-terminus Cys in disulfide bond Asp, Glu, Arg, Lys in salt bridge	An isolated single highly conserved amino acid on the protein surface that is not an expected case.
* Pro is "helix breaking".

Identify the two residues by clicking on them, for example on 4enl you could select Pro290 and Glu188.
Go to the FirstGlance Control Panel and use Find.. to put halos on these two residues. (In the example, you would find "pro290,glu188".)
On your slide, say the two residues you selected. For example

Highly conserved Pro290: expected.
Highly conserved Glu188: unexpected.

Say why you expected conservation of the one residue you chose.
Show a snapshot of the Evolutionary Conservation result for your molecule with halos on the two residues of interest (from Find.). If you used the ConSurf server, paste the address (URL) of your result into your slide!

Section 15 - Cation-pi interaction

List the amino acids (e.g. Phe87:A with Lys212:A) for one energetically significant cation-pi interaction. (":A" means in chain A.)
Show a snapshot of the listed cation-pi interaction with a distance monitor.
Label the amino acids in your snapshot.
Show a snapshot of the CaPTURE report.

Suggestion: FirstGlance, Tools tab, Salt Bridges/Cation Pi

Section 16 - Polyview-3D static image

Show a publication-quality view of your molecule created with Polyview-3D.

Section 17 - Polyview-3D animation for Powerpoint

Show a publication-quality rotating animation of your molecule created with Polyview-3D. If you do this during class, keep the animation small (300 pixels) to avoid overloading the server. Outside of class, 500 or 600 pixels will be plenty large. Animations are large files. You can make the animation file 3x smaller by rocking it instead of doing a full 360 degree rotation. Try rocking through a 60 degree angle in 1 degree steps, with a delay of 5/100 seconds.

Section 18 - Intrinsically disordered regions

If you chose a homology, analyze the TEMPLATE. If you chose a PDB file, analyze that.
Procedure:

In FirstGlance, click on Sequences (in the Molecule Information Tab), and then on the link to the full-length sequence in UniProt.
At UniProt, click on the blue Structure button at the left side of the page.
In the Structure section, look for MobiDB (near the bottom) and click on "Search...".
In MobiDB: in the section titled Predictors, click the green search icon on the Consensus line:
Copy the sequence report and paste it into your slide, as in the example Section 18.

Were any portions of the full-length sequence removed before crystallization? (Refer to Section 4.)
If yes, do they correspond to regions predicted to be disordered?
Are regions predicted to be disordered, but included in the sequence crystallized, missing from the crystallographic model?
Are regions missing from the crystallographic model predicted to be disordered?

ConSurf Server Procedure.
Use this for Section 14:

Go to the ConSurf Server.
Select Amino Acids.
Check YES there is a known protein structure.
Enter the PDB code, or upload your homology model (or biological unit).
Select a chain for analysis. ConSurf can analyze only one chain at a time.
Check NO, you will not be uploading a multiple sequence alignment. (ConSurf will create one for you.)
Leave all parameters at their pre-set defaults. (If you want a research grade result, check "Let me select the sequences for the analysis manually out of BLAST results", and see Limiting ConSurf Analysis to Proteins of a Single Function.)
Enter your email address at the bottom of the form (this is important so you don't lose your results). Optionally enter a job title.
Click the button Submit. It may take an hour or more to complete this calculation.
When it is finished, click the button to view the results in FirstGlance.
Include the web address of the ConSurf result in your report!